Ebook Plant biology and biotechnology (Volume II: Plant genomics and biotechnology): Part 1

Part 1 of ebook Plant biology and biotechnology (Volume II: Plant genomics and biotechnology) provide readers with content about: arabidopsis thaliana - a model for plant research; microalgae in biotechnological application - a commercial approach; application of biotechnology and bioinformatics tools in plant–fungus interactions;... Please refer to the part 1 of ebook for details!

Plant Biology

and Biotechnology

Bir Bahadur · Manchikatla Venkat Rajam

Leela Sahijram · K.V Krishnamurthy

Editors

Volume II: Plant Genomics and Biotechnology

Plant Biology and Biotechnology

Bir Bahadur • Manchikatla Venkat Rajam Leela Sahijram • K V Krishnamurthy

ISBN 978-81-322-2282-8 ISBN 978-81-322-2283-5 (eBook)

DOI 10.1007/978-81-322-2283-5

Library of Congress Control Number: 2015941731

Springer New Delhi Heidelberg New York Dordrecht London

© Springer India 2015

This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software,

or by similar or dissimilar methodology now known or hereafter developed

The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made

Printed on acid-free paper

Bir Bahadur

Sri Biotech Laboratories India Limited

Hyderabad , Telangana , India

Leela Sahijram

Division of Biotechnology

Indian Institute of Horticultural

Research (IIHR)

Bangalore , Karnataka , India

Manchikatla Venkat Rajam Department of Genetics University of Delhi New Delhi , India

K V Krishnamurthy Center for Pharmaceutics, Pharmacognosy and Pharmacology, School of Life Sciences

Institute of Trans-Disciplinary Health Science and Technology (IHST) Bangalore , Karnataka , India

While writing this Foreword, I was reminded of a quote attributed to Mahatma Gandhi: “The expert knows more and more about less and less until he knows everything about nothing.” The quote illustrates the great dilemma that all of

us face in modern times: but this is especially acute for those engaged in the pursuit of science Compared to the times of Archimedes or Leonardo da Vinci or Antonie Philips van Leeuwenhoek, whose range of interests covered several disciplines (they looked at the world in its entirety), most of us have now become narrow specialists of one kind or another, knowing less and less about the wider world Thus, edited monographs, proceedings of seminars and the like have become absolutely essential to keep us informed and engaged in research and teaching more meaningfully (such publications allow summarizing of recent researches at a more advanced level than is pos-sible in ordinary textbooks)

Turning to plant sciences, the Annual Review of Plant Biology, started in

the middle of the last century, continues to be an invaluable source of mation on the broad advances of plant biology Yet, it is necessary to have a more inclusive look at advances over a somewhat longer period and also have this information in a way more organized than the format of annual reviews allows Thus, Prof Bir Bahadur and his colleagues deserve our grateful thanks on undertaking an incredibly diffi cult task of summarizing advances

infor-on the very broad frinfor-ont of plant biology – the topics cover not infor-only tal aspects of plant biology but also plant biotechnology, which is now grow-ing almost as a separate discipline I welcome their style of a historical approach (nearly every article follows this style) This approach is often

fundamen-neglected by specialists, but the fact is that this is the only way to genuine

understanding and for a non-expert to easily discern major advances or stones This unity in overall planning and laying out the style has obviously been possible due to the fact that two of three co-editors are in fact former pupils of the senior editor (Prof Rajam, the senior most of them, was, in a sense, a colleague while I was at Delhi University) Understandably, in the combined work on Volumes I and II, Prof Bir Bahadur is author of nearly ten chapters and Prof Rajam author of fi ve chapters Their two other colleagues

mile-Dr Leela Sahijram and Prof Krishnamurthy have also contributed several chapters Nonetheless, the work has very valuable contributions also from several national and international contributors (in Volume 2, there are around

ten authors from outside India), which has immensely added to the value of

this work

I think that on the whole, a very laudable contribution has been made The

editors have managed to include almost all topics which are signifi cant in

modern plant biology In Volume 1, I was delighted to see several chapters

close to my interest, such as those relating to polyploidy, photosynthesis,

apomixis and fl ower development But in Volume 2, there is special emphasis

on genomics and plant biotechnology, and there are many other chapters of

current interest Space is not adequate to mention all the chapters or their

top-ics, but to me, those on genetic markers, doubled haploids, plant genomes and

genomics (there are several on these topics), epigenetic mechanisms,

bioin-formatics and systems biology were of special interest Also, I am very

delighted that Volume 2 starts with an excellent chapter on Arabidopsis

thali-ana Inspired by a lecture on Langridge’s work by Prof Arthur W Galston, I

undertook in 1960s to ‘tame’ a wild Indian strain of Arabidopsis by raising

in vitro cultures However, despite the fact that Arabidopsis is now the

prin-cipal material for basic research in plant biology, there are many who have

never seen a live Arabidopsis plant, and surely, the opening chapter of this

volume will be valuable for all

Although ably aided by his pupils, Prof Bahadur remains the chief

archi-tect of this endeavour And I am struck with the expanse of his canvas and the

breadth of his interest – it seems to me that in part, it is due to his early

asso-ciation with Prof J.B.S Haldane, F.R.S., whose own interest covered many

disciplines, from mathematics, biochemistry and genetics to animal and plant

biology The topics he and his colleagues cover are of both fundamental and

applied interest I have to admit that many of us in universities are a bit distant

from fi elds and sometimes unfamiliar with the full potential of fundamental

discoveries for biotechnological applications This work will help focus due

attention of readers on both aspects of plant biology

When the chapters were fi rst sent to me, I noticed many typographic

mis-takes than are normally present in fi nished manuscripts – it is true that

Eng-lish is not the mother tongue of many of us in India, but I hope these mistakes

have been rectifi ed

Once again, I wish to congratulate Prof Bir Bahadur and his colleagues

for a very unique monograph and insight in modern plant biology

Honorary Scientist of the Indian, National Satish C Maheshwari

Science Academy, Biotechnology Laboratories

Centre for Converging Technologies

University of Rajasthan , Jaipur , India

The human population is increasing at an alarming rate and is expected to reach 11 billion by 2050 As there is a big gap between population growth and food production, food security for an ever-increasing population poses a major challenge for the present and future times In fact, it will become nec-essary in the coming two decades or so to double food production with avail-able arable land; else, it may precipitate great famines in some parts of the world This is not achievable with just conventional strategies like plant breeding However, the projected increase in food production may be achieved

if traditional breeding methods are coupled with biotechnological approaches

as the latter can offer novel ways for increasing productivity and quality of crops as also for producing an array of useful compounds including pharma-ceuticals and biofuels Indeed, during the past couple of decades, dramatic progress has been made in the fi eld of plant genomics and biotechnology Therefore, a need was felt for updating scientifi c developments in these areas

Plant Biology and Biotechnology – Volume 2 was planned to present

state-of- the-art scientifi c information on various basic and applied aspects of plant genomics This volume comprises 37 chapters spanning various aspects of plant genomics and biotechnology and provides comprehensive and updated

information on a wide variety of topics including Arabidopsis as a wonderful

model system for plant research, plant–fungus interactions, microalgae in biotechnological applications, genetic markers and marker-assisted breeding, doubled haploids in breeding, DNA fi ngerprinting for plant identifi cation, nuclear and organellar genomes, functional genomics, proteomics, epig-enomics, bioinformatics, systems biology, applications of tissue culture in crop improvement and conservation of plant genetic resources, genetically modifi ed crops for production of commercially important products and engi-neering abiotic and biotic stress tolerance, RNAi and microRNAs in crop improvement and environmental, marine, desert and rural biotechnologies The book can serve as a good reference for plant molecular geneticists, plant biotechnologists, plant breeders, agricultural scientists and food scientists Besides, it will also serve as a reference book for post-graduate students, researchers and teachers besides scientists working in agri-biotech companies

Contributors of these volumes were selected from a wide range of tions for introducing a diversity of authors At the same time, these authors were selected based on their vast expertise in specifi c areas of their choice to

institu-match the diversity of topics These authors have a deep understanding of

their subject to enable them not only to write critical reviews by integrating

information from classical to modern literature but also to endure an

unend-ing series of editorial suggestions and revisions of their manuscripts

Need-less to say, this is as much their book as ours

We hope that these books will help our fellow teachers and a generation of

students enter the fascinating world of plant genomics and biotechnology

with confi dence, as perceived and planned by us

Hyderabad , Telangana , India Bir Bahadur

New Delhi , India Manchikatla Venkat Rajam

Bangalore , Karnataka , India Leela Sahijram

Bangalore , Karnataka , India K V Krishnamurthy

First and foremost, we are immensely grateful to all the contributing authors for their positive response We are most grateful to Prof S.C Maheshwari for kindly agreeing to write the Foreword for this volume

We wish to express our grateful thanks to a number of friends and leagues for their invaluable help in many ways and for their suggestions from time to time during the evolution of the two volumes We also thank research scholars of Prof M.V Rajam (University of Delhi South Campus) – Shipra Saxena, Meenakshi Tetorya, Mahak Sachdeva, Bhawna Israni, Mamta, Manish Pareek, Anjali Jaiswal, Jyotsna Naik, Sneha Yogindran and Ami Choubey for their help in many ways

We wish to express our appreciation for the help rendered by Ms Surabhi Shukla, Ms Raman, Mr.N.S Pandian and other staff of Springer for their cooperation and invaluable suggestions Above all, their professionalism, which made these books a reality, is greatly appreciated

We wish to express our grateful thanks to our respective family members for their cooperation

Editors

Bir Bahadur Manchikatla Venkat Rajam

Leela Sahijram K.V Krishnamurthy

1 Arabidopsis thaliana : A Model for Plant Research 1

R Sivasubramanian , Nitika Mukhi , and Jagreet Kaur

2 Microalgae in Biotechnological Application:

A Commercial Approach 27 Nilofer Khatoon and Ruma Pal

3 Application of Biotechnology and Bioinformatics Tools

in Plant–Fungus Interactions 49 Mugdha Srivastava , Neha Malviya , and Thomas Dandekar

4 Genetic Markers, Trait Mapping and Marker-Assisted

Selection in Plant Breeding 65

P Kadirvel , S Senthilvel , S Geethanjali , M Sujatha ,

and K S Varaprasad

5 Doubled Haploid Platform: An Accelerated Breeding

Approach for Crop Improvement 89 Salej Sood and Samresh Dwivedi

6 Plant Molecular Biology Applications in Horticulture:

An Overview 113

Kanupriya Chaturvedi and Leela Sahijram

7 A History of Genomic Structures: The Big Picture 131

Nicolas Carels

8 Organellar Genomes of Flowering Plants 179

Ami Choubey and Manchikatla Venkat Rajam

9 DNA Fingerprinting Techniques for Plant Identification 205

J L Karihaloo

10 Functional Genomics 223

Leonardo Henrique Ferreira Gomes ,

Marcelo Alves- Ferreira , and Nicolas Carels

11 Translating the Genome for Translational Research:

Proteomics in Agriculture 247

Maria Elena T Caguioa , Manish L Raorane , and Ajay Kohli

12 Epigenetic Mechanisms in Plants: An Overview 265

Anjana Munshi , Y R Ahuja , and Bir Bahadur

13 Bioinformatics: Application to Genomics 279

S Parthasarathy

14 Systems Biology: A New Frontier in Science 301

S.R Sagurthi , Aravind Setti , and Smita C Pawar

15 Somatic Embryogenesis 315

Leela Sahijram and Bir Bahadur

16 Micropropagation of Plants 329

Aneesha Singh

17 Efficacy of Biotechnological Approaches to Raise

Wide Sexual Hybrids 347

K R Shivanna and Bir Bahadur

18 Hybrid Embryo Rescue in Crop Improvement 363

Leela Sahijram and B Madhusudhana Rao

19 Applications of Triploids in Agriculture 385

Ashwani Kumar and Nidhi Gupta

20 Improving Secondary Metabolite Production

in Tissue Cultures 397

Ashwani Kumar

21 Somaclonal Variation in Micropropagated Plants 407

Leela Sahijram

22 In Vitro Conservation of Plant Germplasm 417

P E Rajasekharan and Leela Sahijram

23 Gene Banking for Ex Situ Conservation

of Plant Genetic Resources 445

P E Rajasekharan

24 Conservation and Management of Endemic

and Threatened Plant Species in India: An Overview 461

Radhamani Jalli , J Aravind , and Anjula Pandey

25 Biotechnological Approaches in Improvement

of Spices: A Review 487

K Nirmal Babu , Minoo Divakaran , Rahul P Raj ,

K Anupama , K V Peter , and Y R Sarma

26 Metabolic Engineering in Plants 517

Ashwani Kumar

27 Genetically Modified Crops 527

S B Nandeshwar

28 Engineering of Plants for the Production

of Commercially Important Products:

Approaches and Accomplishments 551

Salah E Abdel-Ghany , Maxim Golovkin , and A S N Reddy

29 Genetic Engineering Strategies for Abiotic Stress Tolerance in Plants 579

Francisco Marco , Marta Bitrián , Pedro Carrasco , Manchikatla Venkat Rajam , Rubén Alcázar , and Antonio F Tiburcio

30 Genetic Engineering Strategies for Biotic Stress Tolerance in Plants 611

K Sowjanya Sree and Manchikatla Venkat Rajam

31 RNAi for Crop Improvement 623

Sneha Yogindran and Manchikatla Venkat Rajam

32 Plant MicroRNAs: Biogenesis, Functions, and Applications 639

Manish Pareek , Sneha Yogindran , S K Mukherjee , and Manchikatla Venkat Rajam

33 Environmental Biotechnology: A Quest for Sustainable Solutions 663

Sneha V Nanekar and Asha A Juwarkar

34 Phytoremediation: General Account and Its Application 673

Jitendra K Sharma and Asha A Juwarkar

35 Marine Biotechnology: Potentials of Marine Microbes and Algae with Reference to Pharmacological and Commercial Values 685

M Nagarajan , R Rajesh Kumar , K Meenakshi Sundaram , and M Sundararaman

36 Desert Plant Biotechnology: Jojoba, Date Palm, and Acacia Species 725

Salah E Abdel-Ghany Department of Biology, Program in Molecular Plant

Biology, Program in Cell and Molecular Biology , Colorado State University , Fort Collins , CO , USA

Faculty of Science, Botany Department , Zagazig University , Zagazig , Egypt

Y R Ahuja Department of Molecular Medicine , Vasavi Hospital and

Research Centre , Hyderabad , Telangana , India

Rubén Alcázar Unitat de Fisiologia Vegetal, Facultat de Farmàcia , Universitat de Barcelona , Barcelona , Spain

Marcelo Alves-Ferreira Laboratório de Modelagem de Sistemas Biológicos, National Institute for Science and Technology on Innovation in Neglected Diseases (INCT/IDN) , Centro de Desenvolvimento Tecnológico

em Saúde (CDTS), Fundação Oswaldo Cruz (FIOCRUZ) , Rio de Janeiro , Brazil

K Anupama Division of Crop Improvement and Biotechnology, Indian Institute of Spices Research , Kozhikode , Kerala , India

J Aravind Division of Germplasm Conservation , ICAR-National Bureau of

Plant Genetic Resources , New Delhi , India

Bir Bahadur Sri Biotech Laboratories India Limited , Hyderabad , Telangana ,

India

Lekha Bandopadhyay Bose Institute , Kolkata , West Bengal , India

Marta Bitrián Department of Molecular Mechanisms of Phenotypic Plasticity , Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifi que , Strasbourg , France

Maria Elena T Caguioa Plant Molecular Biology Laboratory , International

Rice Research Institute (IRRI) , Metro Manila , Philippines

Nicolas Carels Laboratório de Modelagem de Sistemas Biológicos, National

Institute for Science and Technology on Innovation in Neglected Diseases (INCT/IDN) , Centro de Desenvolvimento Tecnológico em Saúde (CDTS), Fundação Oswaldo Cruz (FIOCRUZ) , Rio de Janeiro , Brazil

Pedro Carrasco Departament de Bioquímica i Biologia Molecular, Facultat

de Ciències Biològiques , Universitat de València , València , Burjassot , Spain

Kanupriya Chaturvedi Division of Biotechnology , Indian Institute of

Horticultural Research (IIHR) , Bangalore , Karnataka , India

Ami Choubey Department of Genetics , University of Delhi South Campus ,

New Delhi , India

Thomas Dandekar Functional Genomics and Systems Biology group,

Department of Bioinformatics , Biocenter , Wuerzburg , Germany

Minoo Divakaran Indian Institute of Spices Research , Kozhikode , Kerala ,

India

Department of Botany, Providence Women’s College , Kozhikode , Kerala ,

India

Samresh Dwivedi Crop Development-Agri-Business Division , ITC Life

Sciences Technology Centre, ITC-ABD , Secunderabad , Telangana , India

S Geethanjali Centre for Plant Breeding and Genetics , Tamil Nadu

Agricultural University , Coimbatore , Tamil Nadu , India

Maxim Golovkin Foundation for Advancement of Science, Technology and

Research, PA Biotechnology Center , Doylestown , PA , USA

Leonardo Henrique Ferreira Gomes Laboratório de Genômica Funcional

e Bioinformática , Instituto Oswaldo Cruz (IOC) Fundação Oswaldo Cruz

(FIOCRUZ) , Rio de Janeiro , Brazil

Nidhi Gupta Department of Biotechnology , C.C.S University , Meerut ,

Uttar Pradesh , India

Radhamani Jalli Division of Germplasm Conservation , ICAR-National

Bureau of Plant Genetic Resources , New Delhi , India

Asha A Juwarkar Eco-System Division, CSIR-National Environmental

Engineering Research Institute (CSIR-NEERI) , Nagpur , Maharashtra , India

P Kadirvel Directorate of Oilseeds Research , Indian Council of Agricultural

Research , Hyderabad , Telangana , India

J L Karihaloo Asia-Pacifi c Association of Agricultural Research Institutions ,

National Agricultural Science Complex , New Delhi , India

Jagreet Kaur Department of Genetics , University of Delhi South Campus ,

New Delhi , India

Nilofer Khatoon Department of Botany , University of Calcutta , Kolkata ,

West Bengal , India

Ajay Kohli Plant Molecular Biology Laboratory , International Rice

Research Institute (IRRI) , Metro Manila , Philippines

Ashwani Kumar Department of Botany , University of Rajasthan , Jaipur ,

Rajasthan , India

B Madhusudhana Rao Division of Biotechnology , Indian Institute of

Horticultural Research (IIHR) , Bangalore , Karnataka , India

Neha Malviya Department of Biotechnology , Deen Dayal Upadhyay

Gorakhpur University , Gorakhpur , UP , India

Francisco Marco Departament de Biologia Vegetal, Facultat de Farmàcia ,

Universitat de València , València , Burjassot , Spain

K Meenakshi Sundaram Department of Marine Biotechonology , School

of Marine Sciences, Bharathidasan University , Tiruchirappalli , Tamil Nadu , India

S K Mukherjee Department of Genetics , University of Delhi South

Campus , New Delhi , India

Nitika Mukhi Department of Genetics , University of Delhi South Campus ,

New Delhi , India

Anjana Munshi Centre for Human Genetics, School of Health Sciences ,

Central University of Punjab , Bathinda , Punjab , India

M Nagarajan Department of Marine Biotechonology , School of Marine

Sciences, Bharathidasan University , Tiruchirappalli , Tamil Nadu , India

S B Nandeshwar Biotechnology Section , Central Institute of Cotton

Research (CICR) , Nagpur , Maharashtra , India

Sneha V Nanekar Eco-System Division, CSIR-National Environmental

Engineering Research Institute (CSIR-NEERI) , Nagpur , Maharashtra , India

K Nirmal Babu Project Coordinator, All India Coordinated Research Project On Spices (ICAR), Indian Institute of Spices Research , Kozhikode , Kerala , India

Ruma Pal Department of Botany , University of Calcutta , Kolkata , West

Bengal , India

Anjula Pandey Division of Plant Exploration and Germplasm Collection ,

ICAR-National Bureau of Plant Genetic Resources , New Delhi , India

Manish Pareek Department of Genetics , University of Delhi South Campus ,

New Delhi , India

S Parthasarathy Department of Bioinformatics , School of Life Sciences,

Bharathidasan University , Tiruchirappalli , Tamil Nadu , India

Smita C Pawar Department of Genetics , Osmania University , Hyderabad ,

Manchikatla Venkat Rajam Department of Genetics , University of Delhi

South Campus , New Delhi , India

P E Rajasekharan Division of Plant Genetic Resources , Indian institute of

Horticultural Research (IIHR) , Bangalore , Karnataka , India

R Rajesh Kumar Department of Marine Biotechonology , School of Marine

Sciences, Bharathidasan University , Tiruchirappalli , Tamil Nadu , India

Manish L Raorane Plant Molecular Biology Laboratory , International

Rice Research Institute (IRRI) , Metro Manila , Philippines

Muppala P Reddy Center for Desert Agriculture , 4700 King Abdullah

University of Science and Technology , Thuwal , Kingdom of Saudi Arabia

A S N Reddy Department of Biology, Program in Molecular Plant Biology,

Program in Cell and Molecular Biology , Colorado State University , Fort

Collins , CO , USA

S.R Sagurthi Department of Genetics , Osmania University , Hyderabad ,

Telangana , India

Leela Sahijram Division of Biotechnology , Indian Institute of Horticultural

Research (IIHR) , Bangalore , Karnataka , India

Y R Sarma Former Director, Indian Institute of Spices Research , Kozhikode ,

Kerala , India

S Senthilvel Directorate of Oilseeds Research , Indian Council of

Agricultural Research , Hyderabad , Telangana , India

Aravind Setti Department of Genetics , Osmania University , Hyderabad ,

Telangana , India

Jitendra K Sharma Eco-System Division, CSIR-National Environmental

Engineering Research Institute (CSIR-NEERI) , Nagpur , Maharashtra , India

K R Shivanna Conservation Biology, Ashoka Trust for Research in Ecology

and the Environment , Royal Enclave , Bengaluru , Karnataka , India

Samir Ranjan Sikdar Bose Institute , Kolkata , West Bengal , India

Aneesha Singh Discipline of Wasteland Research , CSIR-Central Salt and

Marine Chemicals Research Institute (CSIR-CSMCRI) , Bhavnagar , Gujarat ,

India

R Sivasubramanian Department of Genetics , University of Delhi South

Campus , New Delhi , India

Salej Sood Scientist, Crop Improvement Division, ICAR-Vivekananda

Institute of Hill Agriculture , Almora , Uttarakhand , India

K Sowjanya Sree Amity Institute of Microbial Technology, Amity

University , Noida , India

Mugdha Srivastava Functional Genomics and Systems Biology group,

Department of Bioinformatics , Biocenter , Wuerzburg , Germany

M Sujatha Directorate of Oilseeds Research , Indian Council of Agricultural

Research , Hyderabad , Telangana , India

M Sundararaman Department of Marine Biotechonology , School of

Marine Sciences, Bharathidasan University , Tiruchirappalli , Tamil Nadu , India

Antonio F Tiburcio Unitat de Fisiologia Vegetal, Facultat de Farmàcia ,

Universitat de Barcelona , Barcelona , Spain

K S Varaprasad Directorate of Oilseeds Research , Indian Council of Agricultural Research , Hyderabad , Telangana , India

Sneha Yogindran Department of Genetics , University of Delhi South Campus , New Delhi , India

Prof Bir Bahadur

FLS, C Biol, FI Biol (London)

Dr Bir Bahadur, born 5 April 1938, studied at City College, Hyderabad, for

5 years including an Intermediate Course (Osmania University), graduated from Nizam College and postgraduated from University College, Osmania University, both in the fi rst division He obtained his Ph.D in Plant Genetics from Osmania University He was closely associated with late Prof J.B.S Haldane, F.R.S., a renowned British geneticist who encouraged him to study heterostyly and incompatibility in Indian plants, a subject fi rst studied

by Charles Darwin

He made signifi cant contributions in several areas, especially heterostyly, incompatibility, plant genetics, mutagenesis, plant tissue culture and biotech-nology, morphogenesis, application of SEM in botanical research, plant asymmetry, plant morphology and anatomy and lately the biofuel plants Jat-ropha and castor

He served as Lecturer and Reader at Osmania University, Hyderabad, and

as Reader and Professor at Kakatiya University, Warangal He also served as Head of Department; Chairman, Board of Studies; Dean, Faculty of Science; and Coordinating Offi cer/Dean, UGC Affairs at Kakatiya University He has over 40 years of teaching and over 50 years of research experience He has supervised 29 Ph.D students and 3 M.Phil students in both these universities and has published about 250 research papers/reviews, which are well received and cited in national and international journals, textbooks and reference books

He was a postdoctoral fellow at the Institute of Genetics, Hungarian

Acad-emy of Sciences, Budapest, and worked on mutagenesis and chromosome

replication in Rhizobium He is a recipient of the direct award from the Royal

Society Bursar, London He also worked at Birmingham University (UK) He

was conferred with the title of Honorary Research Fellow by the Birmingham

University He studied species differentiation in wild and cultivated solanums

using interspecifi c hybridization and the enzyme-etched seeds technique in

combination with scanning electron microscopy to assess the relationship

among various Solanum species At the invitation of the Royal Society, he

visited Oxford University, Leeds University, Reading University and London

University, including the Royal Botanic Gardens, Kew, and various research

labs He was invited for international conferences by the US Science

Founda-tion at the University of Missouri, St Louis, at the University of Texas,

Hous-ton (USA), and at the SABRO international conference at Tsukuba, Japan He

has extensively visited most countries of Eastern and Western Europe as well

as Tanzania and the Middle East

He has authored/edited ten books One of his important books is entitled

Jatropha, Challenges for a New Energy Crop , Vol 1 and 2, published by

Springer, New York, USA, 2013, jointly edited with Dr M Sujatha and Dr

Nicolas Carels These books are considered signifi cant contributions to

bio-energy in recent times He was Chief Editor, Proceedings of Andhra Pradesh

Akademi of Sciences , Hyderabad, and Executive Editor, Journal of

Palynol-ogy (Lucknow)

He is the recipient of the Best Teacher Award by the Andhra Pradesh

Gov-ernment for mentoring thousands of students in his teaching career spanning

over 40 years He was honoured with the Prof Vishwambhar Puri Medal of

Indian Botanical Society for his original contributions in various aspects of

plant sciences He has been honoured with the Bharat Jyoti Award at New

Delhi for outstanding achievements and sustained contributions in the fi elds

of education and research He has been listed as one of the 39 prominent

alumni of City College, a premier institution with a long history of about 90

years as per the latest update on its website He has been chosen for

distin-guished standing and has been conferred with an honorary appointment to the

Research Board of Advisors by the Board of Directors, Governing Board of

Editors and Publications Board of the American Biographical Institute, USA

He is a fellow of over a dozen professional bodies in India and abroad

including the following: Fellow of the Linnean Society, London; Chartered

Biologist and Fellow of the Institute of Biology, London Presently, he is an

Independent Director of Sri Biotech Laboratories India Ltd., Hyderabad,

India

Prof Manchikatla Venkat Rajam FNA, FNASc, FNAAS, FAPAS, FABAP

Dr Manchikatla Venkat Rajam is currently Professor and Head, Department

of Genetics, University of Delhi South Campus, New Delhi, India He obtained his Ph.D in Botany (1983) from Kakatiya University, Warangal, India He was a postdoctoral fellow at the prestigious Yale University, New Haven (1984–1985), and also worked at BTI (Cornell University, Ithaca) for

a couple of months as a visiting research associate At Yale University, his work led to the discovery of a new method for the control of fungal plant infections through selective inhibition of fungal polyamine biosynthesis This novel method has been adapted by several research groups globally for the control of a variety of fungal infections, and a large number of research arti-cles have been published in this line of work He returned to India to join as Pool Offi cer (CSIR) and worked for about 2 years (1986–1987) at Kakatiya University Subsequently, he joined the University of Delhi South Campus, where he has been on the faculty since 1987 He had worked in ICGEB, New Delhi, for 6 months as a National Associate of DBT (1994) He made several short visits to various countries including France, Italy, China and Indonesia under the collaborative projects supported by the EU and Indo-French He is

a Fellow of the prestigious Indian National Science Academy (FNA); National Academy of Sciences, India (FNASc); National Academy of Agricultural Sciences (FNAAS); Andhra Pradesh Akademi of Sciences (FAPAS); and Association of Biotechnology and Pharmacy (FABAP) and is an elected member of the Plant Tissue Culture Association, India, since 1995 He was awarded the Rockefeller Foundation Biotech Career Fellowship in 1998 (but could not avail it); the ‘Shiksha Rattan Puraskar’ by the India International Friendship Society in 2011; Department of Biotechnology National Associ-ateship in 1994; and National Scholarship for Study Abroad (Government of India) in 1984 and for Research in 1985 by the Rotary International Club of Hyderabad He is serving as an Associate Editor and member of the editorial board of several reputed journals including BMC Biotechnology and the

OMICS journal Cell and Developmental Biology and is a member of the

advisory or other committees of some universities, institutions as well as other bodies He has guided 28 Ph.D students, 7 M.Phil students and over 22

postdoctoral fellows and has published over 120 papers (80 research articles

in peer-reviewed journals, 15 review articles, 20 book chapters and general

articles) He has one Indian patent to his credit He has vast experience in

plant biotechnology and RNA interference and has handled over 22 major

projects in these areas

Dr Leela Sahijram

Dr Leela Sahijram is currently Principal Scientist, Division of

Biotechnol-ogy, Indian Institute of Horticultural Research (IIHR), Bangalore, India, and

heading the Plant Tissue Culture Laboratory She obtained her M.Sc in Botany

(Plant Physiology) with distinction from Osmania University, Hyderabad, India

(1976), and her Ph.D in Plant Physiology (1983) from the Indian Agricultural

Research Institute, New Delhi, India She was deputed under the USAID

Pro-gram to the University of California at Davis, USA (1992), for plant

transfor-mation She has also undergone training in bioinformatics at IISR, Calicut,

India (2003) She has published several papers in national and international

journals and has guided students for their master’s and doctoral degree

pro-grammes She was identifi ed by the Department of Biotechnology (DBT), New

Delhi, for training on ‘Biotechnology and Intellectual Property Rights (IPR)’ at

the National Law School of India University (NLSIU), Bangalore (2003) She

attended a residential course on ‘Creative Writing in Agriculture’ at the Indian

Institute of Mass Communication (IIMC), New Delhi (2011)

Her team pioneered the micropropagation of banana (globally, the leading

tissue culture–propagated fruit crop), which has spawned a multibillion-

dollar industry worldwide In 1990, she successfully demonstrated over 20

choice clones of banana from across India to be ‘micropropagatable’,

includ-ing cultivars of the Cavendish Group She was member of the Task Force for

the rehabilitation of Nanjangud Rasabale (Pride of Karnataka) syn Rasthali,

‘Silk’ group – a clone threatened with extinction She has also worked

exten-sively on micropropagation and ‘specifi c-pathogen-free’ (SPF) plantlet

pro-duction through meristem culture/micrografting in crops like citrus, caladium,

bougainvillea and chrysanthemum besides bananas and plantains She

spe-cializes in hybrid embryo rescue in perennial horticultural crops

(interge-neric/interspecifi c/intervarietal crosses), particularly in fruit crops, namely,

mango, seedless grapes/citrus, banana and papaya In 2000–2001, she

pio-neered hybrid embryo culture and ex vitro grafting in controlled crosses of

mango

She was conferred with the Dr Vikram Govind Prasad Award 1999–2000 for research on molecular diagnostics of viruses in micropropagated bananas She was also honoured with the Horticultural Society of India Award 2006–

2007 for research on hybrid embryo rescue in seedless grapes and with the Rashtriya Samman Award 2007 for developing biotechnologies for horticul-

tural crops She has been editing the Journal of Horticultural Sciences , an

international journal, for the past 9 years as a Founder Editor She has also

edited a book entitled Biotechnology in Horticultural and Plantation Crops

She has several book chapters in national and international publications to her credit She is the author of many technical and semi-technical popular articles and a laboratory manual besides having trained hundreds of personnel from development departments for setting up commercial plant tissue culture labo-ratories She has travelled widely

Dr K.V Krishnamurthy

Dr K.V Krishnamurthy is currently an Adjunct Professor at the Institute of Trans-Disciplinary Health Science and Technology (IHST), Bangalore, India, and offering consultancy services in Ayurvedic Pharmacognosy He obtained his M.Sc in Botany with University First Rank from Madras University, Chennai, in 1966 and his Ph.D in Developmental Plant Anatomy from the same university in 1973 After a brief stint in government colleges in Tamil Nadu, he joined the present Bharathidasan University, Tiruchirappalli, in

1977 and became a Full Professor in 1989 He has an overall teaching and research experience of more than 47 years and has guided 32 Ph.D scholars, more than 50 M.Phil scholars and hundreds of master’s degree holders He

has published more than 180 research papers and 25 books including

Meth-ods in Cell Wall Cytochemistry (CRC Press, USA) and a textbook on

biodi-versity (Science Publishers, USA), Bioresources of Eastern Ghats: Their

Conservation and Management (with Bishen Singh Mahendra Pal Singh,

Dehradun) His major research areas include plant morphology and genesis, biodiversity, wood science, cytochemistry, plant reproductive

morpho-biology and ecology, tissue culture and herbal medicine and pharmacognosy

He has operated more than 15 major research projects so far He has been a

Fulbright Visiting Professor at the University of Colorado, Boulder, in 1993

and has visited and lectured in various universities in the UK in 1989 His

outstanding awards and recognitions include the following: INSA Lecture

Award 2011; Prof A Gnanam Endowment Lecture Award 2010; President

2007, Indian Association for Angiosperm Taxonomy; Prof V Puri Award

2006 by the Indian Botanical Society; Rashtriya Gaurav Award 2004 by India

International Friendship Society, New Delhi; Scientist of the Year Award

2001 by the National Environmental Science Academy, New Delhi; Tamil

Nadu State Scientist Award 1997–1998 in the Field of Environmental

Sci-ence; Dr V.V Sivarajan Gold Medal Award by the Indian Association for

Angiosperm Taxonomy for Field Study in the year 1997–1998; Prof Todla

Ekambaram Endowment Lecture Award, Madras University, 1997; Prof

G.D Arekal Endowment Lecture Award, Mysore University, 1997–1998;

Prof V.V Sivarajan Endowment Lecture Award, Calicut University, 1997;

Prof Rev Fr Balam Memorial Lecture Award, 1997; 1984 Prof Hiralal

Chakraborty Award instituted by the Indian Science Congress in recognition

of the signifi cant contributions made to the science of botany, 1960; Dr Pulney

Andy Gold Medal awarded by Madras University as University First in

M.Sc Botany, 1966; Dr Todla Ekambaram Prize awarded by Madras

Univer-sity for standing fi rst in M.Sc Plant Physiology, 1966; Maharaja of

Vizianag-aram Prize awarded by Presidency College, Madras, for outstanding

postgraduate student in science, 1965–1966; and Prof Fyson Prize awarded

by Presidency College, Madras, for the best plant collection and herbarium,

1965–1966 He has been the following: Fellow of the National Academy of

Sciences of India (FNASc); Fellow of the Linnean Society, London (FLS);

Fellow of the Indian Association for Angiosperm Taxonomy (FIAT); Fellow

of the International Association of Wood Anatomists, Leiden; Fellow of the

Plant Tissue Culture Association of India; and Fellow of the Indian Botanical

Society He has been the Editor and editorial member of many journals in and

outside India and has also been reviewer of research articles for many

jour-nals He has also served in various committees, the major funding

organiza-tions of India and several universities of India He has been the Registrar and

Director, College and Curriculum Development Council; Member of

Syndi-cate and Senate; Coordinator of the School of Life Sciences and

Environmen-tal Sciences; Head of the Department of Plant Sciences; and a Visiting

Professor in the Department of Bioinformatics at Bharathidasan University,

Tiruchirappalli, before assuming the present job after retirement

Bir Bahadur et al (eds.), Plant Biology and Biotechnology: Volume II: Plant Genomics

and Biotechnology, DOI 10.1007/978-81-322-2283-5_1, © Springer India 2015

Abstract

Arabidopsis thaliana , a small, fl owering, self-pollinating weed, has been

developed into an elegant model system Concerted effort from the plant research community has led to development of extensive genomic resources, tools, and techniques Advances in high-throughput (omics-

based) approaches and their application in Arabidopsis research have

pro-vided ample understanding of basic biological processes in plants Further, bioinformatics platforms allow for integration of the multiple “omics” data, thus, enhancing our appreciation of biological interactions at an

organismal level Taken together, A thaliana has emerged as an excellent

reference source for functional and comparative genomic analysis In this

chapter, we summarize advances made in the fi eld of Arabidopsis research

and resources, tools, and technologies available to the plant scientifi c munity In addition, we briefl y discuss ways in which knowledge gained from this model system can be harnessed for effective deployment in crop improvement

Keywords

Arabidopsis thaliana • Model organism • Forward and reverse genetics •

Functional genomics • Community resources • Crop plants • Plant biology

Department of Genetics , University of Delhi

South Campus , Benito Juarez Road ,

New Delhi 110 021 , India

1

Arabidopsis thaliana : A Model

for Plant Research

R Sivasubramanian , Nitika Mukhi , and Jagreet Kaur

Weed” to a “Model Plant”

Mendel’s seminal work on Pisum sativum (pea)

and, later, Zea mays (maize) brought the two plants into the main foray as ideal systems for studying crop genetics Maize, a major crop plant suitable for cytogenetic studies, played an instru-mental role in providing valuable insights into

various facets of plant biology Horticultural

plants like tomato ( Solanum lycopersicum ) and

petunia ( Petunia hybrida ) were the other models

being used by plant geneticists for studying

bio-logical processes Despite being extensively used

in plant biology, these crops failed to develop

into ideal model systems for studies on molecular

genetics A major drawback with most of these

crop plants being their long generation time and

complex genomes Arabidopsis thaliana , a dicot

fl owering weed belonging to the Brassicaceae

family, was not given much importance until

Friedrich Laibach included it in his search to

identify a plant which had fewer numbers of

large chromosomes suitable for cytogenetic

anal-ysis But, due to the small-sized chromosomes,

Arabidopsis was left out and was not mentioned

in plant research for long (Meyerowitz 2001 )

Laibach refocused his attention on Arabidopsis

in 1943 and proposed it as a genetic model owing

to its short generation time, small size, large

progeny, and self-pollinating lifestyle with

pos-sibility of outcrossing (Fig 1.1 ) Laibach, along

with Albert Kranz, further contributed to

Arabidopsis research by collecting a large number

of natural accessions (750) from around the world George Redei, another plant geneticist, extensively worked towards standardizing muta-

genesis protocols for Arabidopsis and generated

a collection of X-ray induced mutants Langridge,

in 1955, described the fi rst auxotrophic mutant in

higher plants Thereafter, the use of Arabidopsis

mutants to dissect physiological and biochemical pathways underlying various biological pro-cesses gained momentum Maarten Koornneef’s group at Wageningen Agricultural University in Netherlands also started using Arabidopsis

mutants in a major way and constructed its detailed genetic map, further facilitating research

in Arabidopsis genetics (Koornneef and Meinke

2010) Around the same time, Estelle and Somerville ( 1986 ) used Arabidopsis mutants to

characterize important biochemical processes like photorespiration, further emphasizing use-fulness of this plant in genetic analysis Pruitt and Meyerowitz ( 1986) demonstrated that

Arabidopsis had a small genome relative to other

crop models, thereby making mapping and gene cloning comparatively convenient The next big

step in Arabidopsis research was the discovery of

Fig 1.1 ( a ) Large number of seeds can be grown on 90 mm petri plate ( b ) Rosette of 4-week-old Arabidopsis ( c ) a 5-week-old Arabidopsis plant with the infl orescence fl owers

a simple, convenient Agrobacterium tumefaciens -

mediated transformation of germinating seeds

which opened the fl oodgates for developing

vari-ous tools for genetic analysis (Feldmann and

Marks 1987) Clough and Bent ( 1998 ) further

simplifi ed plant transformation by devising the

“fl oral dip” method All these advances, together,

brought this weed into limelight as a model plant

in the fi eld of plant genetics (Fig 1.2 )

“Catalyst” for Plant Research

A relatively smaller genome size (approx

125 Mb) was the simple reason Arabidopsis was

chosen as a subject for the fi rst plant genome

sequencing project By contrast, the genome size

of related Brassica napus (rapeseed mustard) and

Brassica juncea (Indian mustard) is about ten

times that of Arabidopsis Similarly, the genome

of important cereals like rice, maize, and wheat is

much more complex and roughly about 3×, 45×,

100×, respectively, compared to Arabidopsis It

was the fi rst plant genome to be completely

sequenced in the year 2000 under an international

Arabidopsis Genome Initiative (AGI) Analysis

of the genome using various gene-fi nding

algo-rithms, along with supporting data from vast

experimental evidences like EST sequences, MPSS tags, cDNA clones, etc predicts about 33,000 gene models (TAIR 10) The genome analysis also revealed that it is enriched for genes with an average size of 5 Kb (Bevan et al 1998 )

It was also observed that there is very little tive DNA compared to any other higher plant, which facilitates molecular studies and map- based cloning Release of its genome sequence acted as a catalyst for commencement of various projects on functional genomics, leading to gen-eration of a vast stockpile of resources discussed hereunder (Fig 1.2 )

for Functional Genomics

Properties of a living organism are determined mainly by its genetic constitution and its interac-tion with the environment With the ever- expanding wealth of genomic data produced by genome sequencing projects, the next essential step is to decipher the gene function Multiple tools and techniques have been developed for

Arabidopsis with a focus on dissecting and defi

n-ing its gene function and interactions in a given biological process (Table 1.1 )

Fig 1.2 Milestones in Arabidopsis research

1.3.1 Forward Genetic Tools

for Functional Analysis

Forward genetic approach is the classical

phenotype- based approach for screening mutants

in a biological pathway or process of interest

Large-scale forward genetic screens have

pro-vided a basis for the discovery of a multitude of

new genes and pathways fundamental to various

aspects of plant biology Gene disruption is the

most robust and direct approach to address the

biological function of a gene Various libraries of

mutants for forward genetics generated in A

thaliana are available as a public resource and are

discussed below

1.3.1.1 EMS Mutagenesis

Ethyl methane sulfonate (EMS), a known and

commonly used chemical mutagen (alkylating

agent), induces point mutations which vary from

complete knockouts to hypomorphic mutations, thus allowing isolation of a series of allelic vari-ants of a given gene (Bowman et al 1991 ) Isolation of weak alleles is advantageous espe-cially when characterizing genes involved in essential cellular functions EMS treatment has been successfully used for generating a high frequency of irreversible, randomly distributed mutations across the Arabidopsis genome (Greene et al 2003 ) To dissect any biological process, a saturated mutagenized population is screened for a desired phenotype, and the classi-cal positional cloning approach is used for identi-fying the causal mutation/gene (Fig 1.3 ) This approach requires the mutant to be crossed to an

Arabidopsis accession signifi cantly polymorphic

at the DNA level to generate a segregating F 2 mapping population The mapping is done in a biphasic manner, where coarse mapping with a few, well-dispersed genome-wide markers is fol-

Fig 1.3 An overview of gene identifi cation methodology in A thaliana

lowed by fi ne mapping with a large number of

region-specifi c markers A large number of

molecular markers have been used for mapping

polymorphism (SSLP), insertions and deletions

(Indels), cleaved amplifi ed polymorphic

sequences (CAPS), and single nucleotide

poly-morphisms (SNPs) Mapping resolution depends

upon (i) the number of molecular markers

employed and (ii) the number of meiotic

recom-binants analyzed Mutations that interfere with

pattern formation during embryogenesis (Souter

and Lindsey 2000 ), branching pattern (Schmitz

and Theres 1999 ), fl ower morphology (Komaki

et al 1988 ), fl owering time (Putterill et al 1995 ),

response to hormones, and many

cellular/physi-ological processes have been identifi ed in simple,

forward EMS mutant screens With the advent of

“next generation sequencing” (NGS), the

tradi-tional positradi-tional-cloning approach can be

replaced by direct identifi cation of mutations by

whole-genome sequencing Multiple studies have

successfully used NGS to identify directly the

mutation of interest (Schneeberger et al 2009 ;

Austin et al 2011; Schneeberger and Weigel

2011 ; Uchida et al 2011 )

1.3.1.2 Insertional Mutagenesis and Its

Modifi cation

Since the classical positional cloning is an

exten-sive and a long-drawn exercise for identifying a

corresponding gene responsible for a phenotype

of interest, insertional mutagenesis – an alternate

tool for gene disruption – was developed

Integration/insertion of T-DNA/transposable

ele-ment (TE) into the genic region causes disruption

of the gene Since the mutant genes are tagged

with T-DNA inserts, the gene can be easily

iden-tifi ed by isolating the sequences fl anking the

insertion sites Success of this approach depends

upon (i) an easy and effi cient transformation

system and (ii) the ability of T-DNA and

trans-posable elements to integrate randomly into the

host genome (Galbiati et al 2000 ) Effi cient and

simplifi ed Agrobacterium -mediated fl oral dip

transformation method has helped in generating

exceptionally large numbers of insertional

mutants and a near-saturation mutagenesis of the

Arabidopsis genome (Clough and Bent 1998 ) Four major T-DNA mutant collections available

at TAIR collectively encompass 95 % of dicted Arabidopsis genes: (a) SALK lines (Alonso et al 2003 ), (b) GABI-Kat lines (Rosso

pre-et al 2003 ), (c) Syngenta Arabidopsis Insertion Library (SAIL) (Sessions et al 2002 ), and (d) INRA/Versailles lines (Samson et al 2002 ) These stocks are in the public domain and are available from ABRC and NASC stock centers These are popular resources for both forward and reverse genetic approaches for functional analy-sis Similar to EMS mutants, insertion mutants too have been used for unraveling molecular mechanisms underlying various biological pro-cesses, viz., meiotic recombination in plants (Reddy et al 2003 ; Kerzendorfer et al 2006 ), embryo development (Stacey et al 2002 ), organ development (Dievart et al 2003 ), and systemic acquired resistance signaling (Maldonado et al

2002 )

1.3.1.2.1 Trap lines

Traditionally, gene identifi cation relied on ruption of a gene function leading to a recogniz-able phenotype But most of the genes in

Arabidopsis and other crop plants are members

of multigene families and can act redundantly, which makes it diffi cult to characterize them using the classical approach In addition, some phenotypic characters are hard to be detected unless the mutated gene is studied in a certain mutant background which reveals its loss-of- function phenotype Yet another class of genes not amenable to classical genetic studies is the ones that function at multiple developmental stages and whose loss of function may lead to lethality at early developmental stages Modifi cation of the insertional mutagenesis tool kit has led to the development of an alternate powerful strategy that permits gene identifi cation based on their expression pattern, thus eliminat-ing the need for a mutant phenotype (Sundaresan

et al 1995 ; Springer 2000 ) The basic principle underlying this strategy is to randomly integrate into the genome a promoterless reporter con-struct (gene/promoter trap) or a reporter construct with a minimal promoter (enhancer trap) close to

the end of the insertional element (T-DNA or

TE) The expression of the reporter gene is

acti-vated when an endogenous cis-acting promoter

or transcriptional enhancer is present at the site of

integration Bacterial uidA encoding for

β-glucuronidase (GUS) is a commonly used

reporter since endogenous β-glucuronidase

activ-ity in plants is absent (Jefferson et al 1987 )

Alternatively, light emitting bacterial protein

(lux) and luciferase (luc) enzyme from the fi re fl y

have been used as reporters for nondestructive

screens Gene traps have been extensively used to

unravel genes involved in various developmental

processes like lateral root formation (Malamy

and Benfey 1997 ), female gametophyte

develop-ment (Springer et al 1995 ), embryo development

(Topping and Lindsey 1997), and fl oral organ

development (Nakayama et al 2005 ) Trap lines

have also been used to identify stress responsive

genes (Alvarado et al 2004 ) Besides gene

iden-tifi cation, several organ-, tissue-, cell-, and stage-

specifi c markers have been identifi ed which are

useful tools in developmental biology studies

Additionally, the promoter traplines provide a

direct access to highly specifi c promoters For

example, to tackle the problem of drought stress

in crop plants engineering stomatal activity is an

attractive idea Guard cells control the infl ux of

CO 2 for photosynthesis and water loss during

transpiration, and the signaling cascade involved

in these responses are well dissected (Schroeder

et al 2001 ) Francia et al ( 2008 ) screened gene

trap and promoter trap lines to isolate stomata-

specifi c genes and promoters for

biotechnologi-cal applications This approach has also been

extended to other crops like rice to identify cell-

type-/tissue-, stage-, and/or conditionally specifi c

regulatory elements (Yang et al 2004 )

1.3.1.3 Natural Variation

and Association Mapping

Extensive genotypic and phenotypic variations

have been documented in natural accessions of A

thaliana Natural variation is the basis for

tradi-tional linkage mapping/quantitative trait loci

(QTL) mapping aimed at identifying genes

gov-erning a trait of interest F 2 populations and

recombinant inbred lines (RILs) have been used

as experimental populations for QTL mapping RILs allow higher mapping resolution as com-pared to F 2 populations Over 60 RIL populations have been developed and are available to the research community through stock centers The wide range of intraspecifi c diversity (wild acces-sions) available in A thaliana makes it well suited for association mapping (Fig 1.3 ) Association mapping is based on linkage disequi-librium (LD) and offers very high resolution in comparison to traditional linkage mapping, since

it takes advantage of historic recombination events accumulated over several generations

Linkage disequilibrium (LD) in Arabidopsis on

an average extends over 5–10 kb, thus offering nearly single-gene resolution (Kim et al 2007b ) Array-based re-sequencing of 20 maximally

diverse natural accessions of A thaliana has led

to development of a genotyping array (AtSNPtile1) containing probe sets for 2,48,584 SNPs (Kim et al 2007b ) Given the small size of the genome (~125 Mb), this array provides, on average, 1 SNP for every 500 bp, suffi cient enough for genome-wide association mapping There have been several reports of genome-wide association studies (GWAS) in A thaliana

(Aranzana et al 2005 ; Atwell et al 2010 ; Brachi

et al 2010 ; Chan et al 2011 ; Nemri et al 2010 ) The SNP chip has been used for genotyping around 1,307 accessions, and the data is available

to the public (Horton et al 2012 ) Several ware and web-based platforms have been developed for GWAS in plants, viz., TASSEL (Bradbury et al 2007), GAPIT (Lipka et al

soft-2012), GEMMA (Zhou and Stephens 2012 ), Matapax (Childs et al 2012), and the GWA- portal (Seren et al 2012 ) A major drawback of association mapping is the confounding caused due to population structure and the consequent increase in number of false positives In addition, identifi cation of epistatic loci continues to be a major challenge in GWAS A new mapping design that combines advantages of classical QTL mapping and association mapping, known

as nested association mapping (NAM), has been pioneered in maize wherein experimental popu-lations derived from crosses of several founder lines are used (McMullen et al 2009 ; Yu et al

2008 ) In Arabidopsis , two such mapping

popu-lations have been developed, viz., AMPRIL

(Arabidopsis Multiparent RIL) populations

(Huang et al 2011) and MAGIC (Multiple

Advanced Generation Intercross) populations

(Kover et al 2009 ) (Fig 1.3 ) The MAGIC

popu-lation is derived from a heterogeneous stock of

19 inter-mated accessions which have been

com-pletely sequenced, and tools required for QTL/

association mapping in these populations are

freely available (Table 1.1 ) Alternatively,

asso-ciation mapping can be combined with QTL

mapping in several independent RIL populations

to retain statistical power and, yet, not

compro-mise on resolution of mapping Though the

array-based re- sequencing effort led to identifi cation of

around 250K SNPs, it also revealed that the

refer-ence accession Col-0 lacks a substantial portion

of genes present in other accessions A 1001

genome project for A thaliana was announced in

2007 to sequence genomes of other accessions

which would contain sequences not present in the

reference genome (Weigel and Mott 2009 )

(Table 1.1 ) This multinational effort would not

only shed light on local polymorphism patterns

and chromosomal- scale differences but be

directly useful in QTL and association mapping

as well Since many of the accessions sequenced

are parents of RIL populations, availability of the

genome sequence may identify polymorphisms

responsible for various QTLs detected so far

Complete-genome sequences will not only help

identify the causal allele directly in GWAS but

also assist in predicting activity differences

between causal alleles and tackling problems of

allelic heterogeneity and rare variants

1.3.2 Reverse Genetic Tools

for Functional Genomics

The genome annotations for Arabidopsis have

been refi ned overtime and there are more than

30,000 genes predicted, but the role of majority

of these genes in various biological processes is

yet to be elucidated This poses a major challenge

and has made it essential to carry out systematic

genome-wide functional analysis As emphasized

above, cloning the genes based on phenotype requires tremendous labor and time; therefore, complementary reverse genetic approaches are developed to directly investigate the gene function

in specifi c pathways of interest Some of the genetic resources commonly used in the Arabidopsis

functional analysis are discussed below (Fig 1.3 )

1.3.2.1 Sequence-Indexed Insertion

Mutants

As a part of the extensive effort to experimentally validate the function of all the predicted genes, high-throughput thermal asymmetric interlaced- PCR (TAIL-PCR) in combination with sequenc-ing has made it possible to index all the insertion mutants available The gene indexed T-DNA mutant library is a valuable resource for several reverse genetic studies The ultimate aim of the project was to identify at least two genetically stable loss-of-function mutations in all the pre-dicted Arabidopsis genes Using simple PCR- based screening, the mutations can be confi rmed and one can select for lines that are homozygous for the mutation Recently, Bethke et al ( 2014 ) attempted to dissect the role of multiple members

of the Arabidopsis pectin methyl esterases (AtPME) (a 66-member gene family), in pattern- triggered immunity and immune responses to

Pseudomonas , Botrytis , and Alternaria

brassici-cola They identifi ed T-DNA mutants in multiple

members of AtPME gene family and analyzed single and combinations of multiple mutations to address their role in plant defense

1.3.2.2 Targeting Induced Local

Lesions in Genome (TILLING)/ EcoTILLING

EMS mutagenesis can create a higher frequency

of broad-spectrum mutations as compared to the insertion mutagenesis approach The major draw-back of EMS is that the eventual cloning of the mutation is tedious Colbert et al ( 2001 ) devel-oped a PCR-based high-throughput strategy for identifying a SNP in the gene of interest from a mutagenized F 2 population TILLING (Targeting Induced Local Lesion in Genome) combines the robustness of random EMS-induced mutagenesis with high-throughput PCR-based screening to

identify mutations in the gene of interest The

PCR products from the gene of interest are

dena-tured and reannealed to form heteroduplex which

is preferentially digested by the mismatch-

specifi c CEL1 endonuclease, and the cleaved

products are analyzed on a denaturing

polyacryl-amide gel Initially, this reverse genetic strategy

was used to identify numerous mutations in

Arabidopsis mutagenized populations (Enns

et al 2005 ) The use of this technique was further

extended for identifying naturally occurring

genetic variations in the available accessions and

has been named as EcoTILLING (Comai et al

2004) A high-throughput and cost-effective

TILLING approach has been employed by the

Arabidopsis TILLING project (ATP) to provide

series of allelic point mutations for the general

Arabidopsis community This strategy can be

extended to any crop plant for functional

genom-ics and even crop improvement (Slade et al 2005 ;

Cooper et al 2013 )

1.3.2.3 RNA Interference (RNAi)

for Targeted Mutagenesis

Although insertional mutagenesis is an effective

method for generating loss-of-function mutants,

the method falters when dealing with lethal

genes or those genes which are functionally

redundant Targeted gene silencing via

anti-sense, co- suppression, posttranscriptional gene

silencing, and most recently RNA interference

(RNAi) has emerged as a powerful alternative

reverse genetic approach for attenuating the

gene expression In this approach, stable

trans-formants expressing double-stranded RNA

(under a constitutive or inducible promoter)

against target genes are generated, and the effect

of knockdown of the gene expression is

ana-lyzed in terms of the phenotype In comparison

to T-DNA mutagenesis, RNAi lines typically

show a wide spectrum of gene expression, from

no reduction to complete shutdown (Waterhouse

and Helliwell 2003) To date, experimental

proof of function for only 10 % of the predicted

genes is available The AGRIKOLA project

(Arabidopsis Genomic RNAi Knockout Line

Analysis) aims to create targeted gene

knock-down lines via RNAi for all the predicted genes

project, about 150–600 bp long gene-specifi c tags (GSTs) have been designed for approx 25,000 genes which were used to construct dsRNA-expressing vectors These RNAi con-structs have been transformed into wild-type

Arabidopsis plants to generate a library of knockdown transformants These knockdown lines will be an invaluable source for determin-

ing the function of individual Arabidopsis genes

and, by extrapolation, function of orthologous genes in other crop plants as well

1.3.2.4 Gain-of-Function Systems

for Functional Analysis

Activation tagging is another addition in the nal of gene identifi cation tools available to the plant scientifi c community This is a popular gain-of-function approach where the overexpres-sion of the gene results in a novel phenotype Gene activation tagging systems have been estab-

arse-lished in Arabidopsis using either T-DNA vectors

or transposon-based vectors carrying multimers

of the 35S CaMV enhancers (Walden et al 1994 ; Weigel et al 2000; Nakazawa et al 2003 )

Agrobacterium - mediated genome-wide random integration of the activation construct results in upregulation of the gene present in the vicinity of the integration site Ectopic upregulation of the gene can result in an observable phenotype Since the gene is tagged, identifi cation by inverse PCR

or TAIL-PCR can be carried out rapidly Activation tagging approach has been instrumental in deci-phering the function of a number of genes includ-ing ADR1 in defense response (Grant et al 2003 ; Aboul-Soud et al 2009 ), BAK1 in brassinosteroid signaling (Li et al 2002 ), and FT in fl oral transition (Kardailsky et al 1999 )

FOX hunting system (full-length sion of cDNA for gene hunting) is an alternative approach to activation tagging where the ease of

overexpres-transformation of Arabidopsis and the

availabil-ity of the full-length cDNA sequences have been exploited As opposed to activation tagging, genes responsible for the overexpression pheno-type can be easily identifi ed Using a normalized full-length cDNA library, Ichikawa et al 2006

have developed 30,000 independent Arabidopsis

FOX lines which are available from RIKEN As a

further extension of this, full-length rice cDNA

clones have been transformed into Arabidopsis

with an aim of screening for rice functional genes

in a high-throughput manner (Sakurai et al

2010 )

Tool for Deciphering Gene

Function

Functional genomics is a genome-wide

approach that attempts to use the ever expanding

wealth of data produced through various

high-throughput analysis for defi ning the gene

func-tion and its interacfunc-tions in a given biological

process

1.4.1 Transcriptomic Resources

for A thaliana

Transcriptomics, a comprehensive study of the

whole-genome expression, is an informative

approach towards functional gene analysis The

spatial and temporal expression of a gene to a

certain extent is refl ective of its activity within

the cell This section discusses the various

publi-cally available resources for gene expression

analysis and their application in functional

genomics

1.4.1.1 Expression Profi ling Provides

Insights into Gene Function

To get a glimpse of the transcriptional activity

within the cell, a large-scale expressed sequence

tag (EST) sequencing project was undertaken in

Arabidopsis The data generated in the EST

sequencing was useful in gene discovery as it

helped in annotating the expressed regions in the

genome besides providing information about the

gene expression Serial analysis of gene

expres-sion (SAGE) and its various modifi cations like

micro SAGE and mini SAGE were commonly

used by various research groups to identify large

number of differentially expressed transcripts

present in different tissues/conditions More

recently, the NGS-based massively parallel signature sequencing (MPSS) has gained the edge In this approach, short sequence tags are generated for a cDNA library by sequencing approx 20–25 bp from the 3′ side of cDNA Besides discovering novel transcripts, MPSS also provides a robust method for assess-ing the transcript abundance Additionally, the MPSS platform has been used to identify a large number of small RNAs

The hybridization-based approaches for acquiring large-scale gene expression profi les

have also been established for Arabidopsis and

are being continuously improved The traditional microarrays used for analyzing the transcrip-tional profi les were biased towards the known and predicted genes With the whole genome sequence available, it became possible to develop tiling arrays (TA) and whole genome arrays (WGA) These arrays cover the entire genome with probes either at regular interval (TA) or probes that are overlapping along the entire length of the genome (WGA) These arrays have not only been used for estimating the transcript levels but also play a signifi cant role in identify-ing novel transcripts, various alternate transcripts, and polymorphisms (Mockler et al 2005 ) Using the WGA, Zeller et al ( 2009 ) studied the stress-

induced changes in the A thaliana transcripts in

response to various abiotic stresses like salt, osmotic, cold, and heat They identifi ed several novel stress-induced genes which were missed in the earlier classical microarray experiments Thousands of microarray experiments that have

been conducted in different laboratories with A

thaliana now form a part of large quantitative

data on gene expression in different tissue and in response to different treatments and experimental conditions Similarly, a comprehensive expres-sion atlas for A thaliana has been developed

based on WGA and is available at A thaliana

Tiling Array Express (At-TAX) (Laubinger et al

2008 ) The utility of the tiling array has been ther extended by combining this platform with immunoprecipitation methods for detecting chro-mosomal locations at which protein-DNA inter-action occurs across the genome (Wang and Perry

fur-2013 ) Further, Zhang et al ( 2006 ) generated an

extensive Arabidopsis methylome data by

coupling tiling arrays with methylcytosine

immunoprecipitation methods Most of the

microarray data has been deposited in the public

databases such as NASC and TAIR, and this data

can be accessed for analysis either directly from

these sites or through the various available links/

tools like Genevestigator (Zimmermann et al

2004) and MAPMAN (Thimm et al 2004 )

(Table 1.1 )

1.4.1.2 Co-expression Analysis: Guilt by

Association

Since an extensive set of microarray expression

data across multiple experiments is available, the

attention has now shifted to exploring the

corre-lated expression of the entire genome with fi ne

focus on defi ning specifi c pathways in a

biologi-cal process These correlation studies are a

pow-erful tool to identify new genes which could be

functionally related Several co-expression

anal-ysis interfaces like ATTED-II and CressExpress

have been developed that use the comprehensive

collection of the publicly available transcriptome

data sets to identify co-regulated genes

(Nakashima et al 2009) The PLAnt co-

Expression database (PLANEX) is a genome-

wide database which includes publicly available

GeneChip data obtained from the Gene

Expression Omnibus (GEO) A comparative

transcriptomic analysis using meta-analysis

approach for drought (abiotic) and bacterial

(biotic) stress response in rice and Arabidopsis

identifi ed several genes, common to both the

stresses (Shaik and Ramakrishna 2013 ) This

identifi cation of master regulatory genes that act

in biotic and abiotic stress response would be the

potential candidates for manipulating stress

toler-ance in crop plants as well

Though co-expression analysis proves to be a

powerful tool to identify new genes which could

be functionally related, one needs to keep in mind

that the co-expression analysis is a refl ection of

regulation at the mRNA level only The analysis

would gain weightage if any known

protein–pro-tein interaction information can be integrated

into the co-expression analysis

1.4.1.3 Small RNA Database and Tools

for Functional Genomics

The regulation of gene expression occurs at multiple levels including mRNA stability It has become evident that small RNAs are one of the major players in regulating gene expression during growth, development, and stress responses in plants The cost-effective next generation sequencing has made it possible to discover hundreds of small RNA from

Arabidopsis and other plants This extensive data is available through the web interface –the Arabidopsis Small RNA Project (ASRP) which also integrates the community- wide resources related to small RNA and various bioinformatic tools for mi- and siRNA identifi cation (Backman et al 2008 ) The Arabidopsis model

system has been useful in dissecting the small RNA component of genetic and epigenetic reg-ulation in plant development, growth, and dis-ease resistance Palatnik et al ( 2003 ) showed the involvement of microRNA in controlling leaf morphogenesis Auxin signaling responses were also shown to be regulated via small RNA Several auxin response factors (ARFs) were predicted to be targets for miRNA ARF10 and ARF17 contain potential sites for miR160 (Jover-Gil et al 2005) Overexpression of miR160 resistant version of ARF17 led to higher accumulation of ARF17 These changes

in expression correlate with the pleotropic phological abnormalities and reduced fertility observed in the transgenic suggesting the regu-lation of ARF17 by miR160 (Mallory et al

mor-2005 ) Similarly, another miR393 also plays a signifi cant role in integrating the environmental cues to auxin signaling pathway (Windels and Vazquez 2011 )

Additionally, using the basic principle of small RNA-directed gene silencing, virus induced gene silencing (VIGS), hairpin-based RNA interference (RNAi), and artifi cial microRNA (amiRNA) tools have been developed

to regulate targeted gene expression The use of these strategies has been further extended for selectively regulating gene expression in crop plants as well

1.4.1.4 Tools for Regulatory Sequence

Analysis

The control of gene expression is pivotal to all

cellular processes, and one of the major

chal-lenges in biology is to unravel the mechanisms

that regulate gene expression The gene function

is directly linked to its spatial and temporal

expression which is regulated by a network of

transcription factors, the key regulatory proteins

The cues for gene regulation are hard wired into

the promoter region which is formed by

cis-regu-latory elements These cis-regucis-regu-latory elements

are recognized by specifi c transcription factors

Therefore, in order to understand the gene

expres-sion and thereby the gene function, basic

infor-mation on the transcription factors and their

binding sites is important A thaliana encodes

more than 1,500 transcription factors which are

classifi ed into 40–50 families based on the

sequence similarity (Riechmann et al 2000 )

Arabidopsis Gene Regulatory Information Server

(AGRIS) is the interface that hosts AtcisDB,

AtTFDB, AtRegNet, and ReIN databases

(Table 1.1 ), which provide a catalogue of cis ad

trans factors involved in gene regulation

1.4.2 Epigenomic Resources

Epigenetics is the study of changes in the

regula-tion of gene expression that do not involve a

change in the DNA sequence Epigenetic changes/

modifi cations include methylation of the DNA,

chemical modifi cation of the histones, and

alter-native histone variants These modifi cations are

known to play important roles during

develop-ment and in responses to different environdevelop-mental

cues An integrated epigenome map of Arabidopsis

was published in 2008, which describes

interac-tions between the methylome, transcriptome and

the small RNA transcriptome, and their effect on

gene regulation (Lister et al 2008 ) Further, the

epigenetic variation between the different

acces-sions of Arabidopsis and its dependence on the

genetic variation between the accessions has been

reported (Schmitz et al 2013 ) DNA methylations

and their effect on the transcriptional activity have been studied in response to biotic stress (Dowen et al 2012 ) The Epigenomics of Plants International Consortium (EPIC) provides a list

of epigenetic resources in the form of databases and tools, which are available to the research community (Table 1.1 ) Also, The EPIC-CoGe Epigenomics Webbrowser provides a user-friendly interface to scan and search for epigen-etic marks in the genome (Table 1.1 )

1.4.3 Proteomics Resources

Transcriptional analysis in Arabidopsis using

the whole genome arrays and tiling arrays gested that the transcriptional capacity of the

Arabidopsis genome is far more than predicted

by genome annotation Many non-annotated intergenic regions were transcribed and a lot of antisense transcripts were identifi ed These fi nd-ings prompted the scientists to look at the global translational products and their posttranslational modifi cations which are known to play a key role in many cellular processes like cell signal-ing, regulation of gene expression, protein degradation, and protein–protein interactions

“Proteomics” is the study and characterization

of the complete set of proteins present at a given time in the cell and organelle using gel- and mass spectrometry-based high-throughput tech-

niques Proteome profile of Arabidopsis has

led to the identifi cation of several organ-specifi c biomarkers for different developmental stages, organs, and undifferentiated cell cultures (Baerenfaller et al 2008 ) A proteome map of

different root cell types using Arabidopsis root

marker lines and fl uorescence activated cell sorting (FACS) was recently released (Petricka

et al 2012 ) Proteome profi ling of Arabidopsis

under diverse abiotic stresses such as cold, drought, salinity, hypoxia, etc (Kosova et al

2011 ; Ghosh and Xu 2014 ) determined the role

of several families of transcriptional factors (TFs) and protein phosphorylation/dephosphor-ylation events in mediating stress response For